Although gene therapy technology is becoming more advanced and sophisticated there are still a number of technical hurdles which limit the usefulness of this technology. One of the important technical hurdles pertains to the difficulty of delivering DNA into a cell and having that DNA reach its intended target so that genetic transformation can occur. There are several steps in this process including finding an appropriate vehicle for delivering the DNA, increasing the efficiency by which DNA enters the cell, and increasing the likelihood that the DNA will be released by the delivery vehicle so that it reaches its intended location within the cell.
The use of cationic organic molecules to deliver heterologous genes in gene therapy procedures has been reported in the literature. Not all cationic compounds will complex with DNA and facilitate gene transfer. Currently, a primary strategy is routine screening of cationic molecules looking for good candidates. The types of compounds which have been used in the past include cationic polymers such as polyethyleneamine, ethylene diamine cascade polymers, and polybrene. Proteins, such as polylysine with a net positive charge have also been used. The largest group of compounds, cationic lipids; includes DOTMA, DOTAP, DMRIE, DC-chol, and DOSPA. All of these agents have proven effective but suffer from potential problems such as toxicity and expense in the production of the agents.
Cationic liposomes are currently the most popular system for gene transfection studies. Cationic liposomes serve two functions: protect DNA from degradation and increase the amount of DNA entering the cell. While the mechanisms describing how cationic liposomes function have not been fully delineated, such liposomes have proven useful in both in vitro and in vivo studies. However, cationic liposomes suffer from several important limitations. Such limitations include low transfection efficiencies, expense in production of the lipids, low suspenibility when complexed to DNA, and toxicity. Dissociation of DNA from DNA/liposomes complexes is also one of the major barriers for cationic liposome-mediated gene transfection (Rolland, A. P. (1998) Crit Rev Ther Drug Carrier Syst 15:143-198; Escriou, V., C. Ciolina, A. Helbling-Leclerc, P. Wils, D. Scherman (1998) Cell Biol Toxicol 14:95-104; J. Zabner, A. J. Fasbender, T. Moninger, K. A. Poellinger, M. J. Welsh (1995) J Biol Chem 270:18997-19907).
Since Felgner et al. ((1987) Proc Natl Acad Sci USA 84:7413-7417) reported application of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) in transfection of plasmid DNA in 1987 (Felgner P. L. et al. (1987) Proc Natl Acad Sci USA 84:7413-7417), many cationic lipids have been synthesized and used in plasmid DNA delivery (Gao, X, L. Huang (1995) Gene Ther 2:710-722; Rolland, A. P. (1998) Crit Rev Ther Drug Carrier Syst 15:143-198; Lee, R. J., L. Huang (1997) Crit Rev Ther Drug Carrier Syst 14:173-206). Most of these efforts have been focusing on improving the transfection activity and decreasing the cytotoxicity. The uses of ester, amide, and carbamate linkages to tether polar and hydrophobic domains are common strategies to lower the toxicity. However, no intracellular degradation studies have been conducted for any cationic transfection lipid (Aberle, A. M., F. Tablin, J. Zhu, N. J. Walker, D. C. Gruenert, M. H. Nantz (1998) Biochemistry 37:6533-6540). 3-(-[N-(N',N'-dimethyl amino ethane)carbomayl]cholesterol (DC-Chol) was the first lipid used in clinical trials because of its combined properties of transfection efficiency, stability and low toxicity (Gao, X., L. Huang (1995) Gene Ther 2:710-722). Recently, Aberle et al. ((1998) Biochemistry 37:6533-6540) reported a novel tetraester construct that reduced the cationic lipid-associated cytotoxicity compared to DC-Chol (Aberle, A. M., F. Tablin, J. Zhu, N. J. Walker, D. C. Gruenert, M. H. Nantz (1998) Biochemistry 37:6533-6540). However, the introduction of the ester bond may also decreases the stability of liposome in systemic circulation when the liposomes are used in clinical trials.
Compounds containing disulfide bonds are able to participate in disulfide exchange reactions over a broad range of conditions from acid to basic pH and in a wide variety of buffer constitutes and physiological conditions (Hermanson, G. T. (1996) Bioconjugate Techniques, pp. 150-152, Academic Press, San Diego). Because of their special chemical properties, disulfide conjugate techniques have been widely used in drug delivery to achieve high delivery efficiencies (Trail, P. A., D. Willner, S. J. Lasch, A. J. Henderson, S. J. Hofstead, A. M. Casazza, R. A. Firestone, I. Hellstrom, K. E. Hellstrom (1993) Science 261:212-215; Legendre, J. Y., A. Trzeciak, B. Bohrmann, U. Deushle, E. Kitas, A. Supersaxo (1997) Bioconjugate Chem. 8:57-63; Kostina, E. V., A. S. Boutorine (1993) Biochimie 75:35-41; Trail, P. A., D. Willner, J. Knipe, A. J. Henderson, S. J. Lasch, M. E. Zoeckler, G. R. Braslawsky, J. Brown, S. J. Hofstead, R. S. Greenfield, R. A. Firestone, K. Mosure, K. F. Kadow, M. B. Yang, K. E. Hellstron, I. Hellstrom (1997) Cancer Research 57:100-105). The most common method used in bioconjugates involves cross-linking or modification reactions using disulfide exchange processes to form disulfide linkage with sulfhydryl-containing molecules. However, this method is not suitable for the syntheses of most cationic lipids due to their specific chemical structures.
Thus, as can be understood from the above, there remains a need in the art for materials and methods which can be used for the efficient delivery of molecules, such as DNA, into cells. The use of cationic compounds containing disulfide bonds to deliver DNA in gene therapy procedures has not previously been described.